Related Product Information

Introduction

System Overview

The FastTrack® MAG mRNA Isolation Kit uses oligo(dT)-conjugated magnetic beads to isolate poly A+ RNA directly from cell lysate, tissue lysate, or total RNA in 1–1.5 hours using conventional laboratory equipment (Morrissey et al., 1989; Stone et al., 1996). The high quality and quantity of the isolated mRNA makes it suitable for use in a variety of applications, including cDNA library construction, microinjection, in vitro translation, RT-PCR, Northern blotting. The mRNA captured on the magnetic beads may also be used in some applications without elution. Using the kit, you can isolate mRNA from varying amounts of cells (<1 × 105 – 5 × 107 cells), tissue samples (< 10 mg–> 300 mg of tissue), or total RNA (< 1 μg–2000 μg). Separate procedures are provided for each type of sample.

Workflow

Using the FastTrack® MAG mRNA Isolation Kit, you first isolate total RNA using a method of choice, or prepare cells/tissue samples using the reagents provided in this kit. Then you prewash the FastTrack® MAG Beads, bind the sample, and perform a series of wash steps. Finally, you elute the mRNA from the beads using RNase-free water provided in the kit.
Isolate Total RNA Lyse Cells Homogenize tissue 20-30 Minutes
↓ ↓ ↓

Prewash FastTrack® MAG Beads and bind sample 20-30 Minutes
↓

Wash FastTrack® MAG Beads 10-20 Minutes
↓

Elute sample 5-10 Minutes

Advantages of the Kit

The FastTrack® MAG mRNA Isolation Kit offers the following advantages:

Isolates high-quality mRNA in less than 1 hour from total RNA and 1.5 hours from cells or tissue

Isolated mRNA has minimal ribosomal RNA and genomic DNA contamination

Higher yields of mRNA than comparable systems

mRNA may be eluted at a high concentration for specific downstream applications

Compatible with high-throughput applications

Typical Yields

Source

Recovery of mRNA

1 mg total RNA (from mammals)

>30 μg (>3%)

1 mg total RNA (from plants)

>15 μg (>1.5%)

1 × 106 cells

1 μg

1 g tissue

5–80 μg*

*Yields of mRNA from tissue are highly dependent on the type of tissue.

Magnetic Particle Separator

This kit requires the use of a magnetic particle separator (MPS) with holes for 1.5-ml tubes to separate the FastTrack® MAG beads in solution. The Magna-Sep™ MPS is a 6-hole magnetic particle separator available from Invitrogen that can hold 1.5–2.0-ml tubes (Catalog no. K1585-01). You may also use separators from other manufacturers.

Note: If the total RNA contains high-level of genomic DNA or you are performing downstream RT-PCR, perform a DNase I digestion step to minimize genomic DNA contamination prior to mRNA purification.

Amounts of Beads and Buffers

The following table lists the amounts of FastTrack® MAG Beads, Binding Buffer B6, and Wash Buffer W7 to use with the specified amount of total RNA. Note: The isolation procedure uses six separate volumes of Wash Buffer W7, so each volume is shown × 6.

Amount

Micro

Maxi

Total RNA

< 1 μg

1–50 μg

50–500 μg

500–2000 μg

FastTrack® MAG Beads

20 μl

50 μl

100 μl

≥ 200 μl*

Binding Buffer B6

100 μl

200 μl

500 μl

500 μl

Wash Buffer W7

100 μl × 6

200 μl × 6

500 μl × 6

500 μl × 6

*To ensure maximum mRNA yield, the ratio of total RNA (μg) to beads (μl) should be > 7.5 (i.e., at least 200 μl of beads per 1500 μg of total RNA).

Before Proceeding

Before proceeding with the protocol, preheat the Binding Buffer B6 and add water to the total RNA sample as described below. While the buffer is heating, proceed with the Prewash and Binding Procedure above.

Add the volume of Binding Buffer B6 specified in the table on the previous page to a 1.5-ml RNase-free tube. Place the tube in a heat block or thermal cycler at 65-70°C.

In a separate 1.5-ml RNase-free tube, add RNase-free water to your total RNA sample to a total volume equal the volume of Binding Buffer B6 in Step 1. Place the sample tube on ice.

Important: When following the procedures in this section, be careful to never let the beads dry out.

Pipetting Liquid from Tubes

When pipetting liquid from a tube in the magnetic separator, always point the pipette tip toward the opposite side of the tube bottom from the pellet to avoid touching the beads.

Prewash and Binding Procedure

While the Binding Buffer B6 is preheating (see previous page), proceed with the steps below:

Thoroughly resuspend the FastTrack® MAG Beads by pipetting them gently up and down. Transfer the amount of resuspended beads specified in the table on page 4 to an RNase-free microcentrifuge tube.

Insert the microcentrifuge tube into the magnetic particle separator (MPS). When the beads are clearly separated from the liquid (∼0.5–2 minutes), pipette the liquid out of the tube and discard, and immediately add the volume of Wash Buffer W7 specified in the table on page 4. Important: Do not let the beads dry out.

Remove the tube from the magnetic particle separator and resuspend the beads by pipetting gently up and down.

Repeat Steps 2–3 one more time, and then proceed to Step 5.

Insert the tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), pipette the liquid out of the tube and discard, and immediately add the total RNA sample from Step 2 above and the heated Binding Buffer B6 from Step 1 above.

Remove the tube from the magnetic separator and resuspend the beads in the solution by pipetting gently up and down. (Note: Pipetting too vigorously may damage the RNA.)

Cap the tube and place in the heat block or thermal cycler. Incubate at 65–70°C for 2–5 minutes.

Transfer the tube to a rotator and rotate the sample for 10 minutes at room temperature. Proceed to Wash and Elution Procedure below.

Wash and Elution Procedure

Insert the tube from Prewash and Binding Procedure, Step 8, in the magnetic separator. When the beads are clearly separated (∼0.5–2 minutes), remove and save the supernatant. Note: For rare samples, if your yield is low, you may want to preserve this supernatant and attempt to perform a separate isolation with it.

To the beads, immediately add the volume of Wash Buffer W7 specified in the table

Remove the tube from the magnetic separator and resuspend the beads in the wash buffer by pipetting gently up and down.

Reinsert the tube in the magnetic separator. When the beads are clearly separated (∼0.5–2 minutes), remove the wash buffer from the tube and discard. Be careful to completely remove the wash buffer.

Repeat Steps 2–4 three more times, and proceed to Step 6.

If you started with < 50 μg of total RNA, immediately add 5-20 μl of RNase-free water. If you started with > 50 μg of total RNA, immediately add 20–50 μl of RNase-free water.

Remove the tube from the magnetic separator and thoroughly resuspend the beads by pipetting gently up and down. Incubate at 37°C for 2–5 minutes. Use the longer incubation time for larger samples.

Insert the tube into the magnetic separator. When the beads are clearly separated (∼0.5–2 minutes), remove and save the supernatant. Important: The supernatant contains the isolated mRNA. Do not discard.

Repeat Steps 6–8, using 5 μl of RNase-free water for < 50 μg of total RNA and 10 μl of RNase-free water for > 50 μg of total RNA. Again, save the supernatant.

Combine the supernatants from Steps 8 and 9. This is your isolated mRNA. Store mRNA at -80°C.

The mRNA quality and yield may be determined by spectrophotometry and agarose gel electrophoresis, as described above.

To the cell pellet in a microcentrifuge tube, add the Lysis Buffer solution from Step 2, above.

For <1 × 105 cells, pipette the lysate up and down at least 10 times. For >1 × 105 cells, shear the DNA by passing the lysate through a 21-gauge needle fitted to a 1-ml syringe. Pass the lysate through the needle ≥20 times for Micro kit volumes and ≥30 times for Maxi kit volumes.

Transfer the tube to a microcentrifuge and spin at maximum speed for 5 minutes at room temperature.

Incubate the sample at 45°C in a water bath or incubator for 10 minutes. While the sample is incubating, proceed to Prewash and Binding Procedure below.

Pipetting Liquid from Tubes

When pipetting liquid from a tube in the magnetic separator, always point the pipette tip toward the opposite side of the tube bottom from the pellet to avoid touching the beads.

Important: When following the procedures in this section, be careful to never let the beads dry out.

Prewash and Binding Procedure

While the lysate is incubating (above), proceed with the steps below:

Thoroughly resuspend the FastTrack® MAG Beads by pipetting them gently up and down. Transfer the amount of resuspended beads specified in the table above to an RNase-free microcentrifuge tube.

Insert the microcentrifuge tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), pipette the liquid out of the tube and discard, and immediately add the volume of Wash Buffer W7 specified in the table above. Important: Do not let the beads dry out.

Remove the tube from the magnetic separator and resuspend the beads in the wash buffer by pipetting gently up and down.

When the incubation time of the lysate has ∼4 minutes left (Step 5), repeat the wash procedure in Steps 2 and 3 above one more time, and then proceed to Step 5.

Insert the tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), pipette and discard the solution, and immediately add the lysate from Cell Lysis, Step 5, above, and the heated Binding Buffer B6 from Preparing the Binding Buffer and Lysis Buffer, Step 1.

Remove the tube from the magnetic separator and resuspend the beads completely in the solution by pipetting up and down.

Cap the tube and place in the heat block or thermal cycler. Incubate at 65–70°C for 2–5 minutes.

Transfer the tube to a rotator and rotate the sample for 10 minutes at room temperature. Proceed to Wash and Elution Procedure below.

Wash and Elution Procedure

Insert the tube from Prewash and Binding Procedure, Step 8, above, in the magnetic separator. Visually inspect the beads until they are clearly separated (∼2–5 minutes). Note: Beads may take longer to separate depending on the viscosity of the lysate and the abundance of non-sheared genomic DNA.

When the beads are separated, remove and save the supernatant. Note: For rare samples, if your yield is low, you may want to preserve this supernatant and attempt to perform a separate isolation with it.

To the beads, immediately add the volume of Wash Buffer W6 specified in the table on above.

Remove the tube from the magnetic separator and resuspend the beads by pipetting gently up and down.

If you are using > 1 × 106 cells and/or performing downstream RT-PCR: Add 1 μl (1 unit) of DNase I, Amplification Grade, per 100 μl of Wash Buffer W6 to the tube. Mix by pipetting gently up and down and incubate at 25°C for 5–10 minutes.

Reinsert the tube in the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), remove the wash buffer from the tube and discard. Carefully remove the wash buffer completely.

To the beads, immediately add the volume of Wash Buffer W7 specified in the table above.

Remove the tube from the magnetic separator and resuspend the beads by pipetting gently up and down.

Reinsert the tube in the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), remove the wash buffer from the tube and discard. Be careful to completely remove the wash buffer.

Repeat Steps 7–9 using Wash Buffer W7 two more times, then proceed to Step 11.

If you started with < 1 × 106 cells, immediately add 5-20 μl of RNase-free water. If you started with > 1 × 106 cells, immediately add 20–50 μl of RNase-free water. Remove the tube from the magnetic separator and thoroughly resuspend the beads by pipetting gently up and down. Incubate at 37°C for 2–5 minutes. Use the longer incubation time for larger samples.

Insert the tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5-2 minutes), remove and save the supernatant.

Important: The supernatant contains the isolated mRNA. Do not discard.

The following table lists the amounts of FastTrack® MAG Beads, Lysis Buffer, Proteinase K, Binding Buffer B6, Wash Buffer W6, and Wash Buffer W7 to use with the specified amount of tissue.
Note: The isolation procedure has five separate washes using Wash Buffer W7, so each volume is shown × 5.

Amount

Micro

Maxi

Tissue

< 10 mg

10–50 mg

50–200 mg

<200–500 mg*

FastTrack® MAG Beads

20 μl

20-50 μl

50-100 μl

100-200 μl

Lysis Buffer L4

100 μl

200 μl

500 μl

500 μl

Proteinase K

2.5 μl

5 μl

10 μl

10 μl

Binding Buffer B6

100 μl

200 μl

500 μl

500 μl

Wash Buffer W6

100 μl

200 μl

500 μl

500 μl

Wash Buffer W7

100 μl × 5

200 μl × 5

500 μl × 5

500 μl × 5

*For tissue amounts > 200 mg, you can use more Lysis Buffer L4/Proteinase K and Binding Buffer B6 in multiple tubes or proportionally scale up to a larger tube.

The abundance of mRNA varies greatly in different tissues. Adjust the volumes accordingly.

To maximize the yield of mRNA from tissues, we suggest first isolating total RNA from the tissue using TRIzol® Reagent or another method and then isolating mRNA from the total RNA using this kit

Preparing Binding Buffer and Lysis Buffer

Preheat the Binding Buffer B6 and prepare the Lysis Buffer L4 as described below:

Add the volume of Binding Buffer B6 specified in the table above to an RNase-free tube. Place the tube in a heat block or thermal cycler at 65–70°C.

In a separate RNase-free tube, add the volume of Proteinase K specified in the table above to the specified volume of Lysis Buffer L4. Proceed with Preparing the Tissue Sample.

Important: Homogenize tissue in the presence of lysis buffer to ensure immediate inactivation of any RNases that are released as the cells lyse. Complete homogenization is critical for complete cell lysis and inactivation of RNases.

Before use, clean and wash the homogenizer tip and then autoclave and bake for 3 hours or overnight at 210°C.

Pipetting Liquid from Tubes

When pipetting liquid from a tube in the magnetic separator, always point the pipette tip toward the opposite side of the tube bottom from the pellet to avoid touching the beads.

Preparing the Tissue Sample

Prepare the tissue sample as described below.

Add the Lysis Buffer L4 plus Proteinase K from Step 2, above, to your frozen or fresh tissue sample in a sterile microcentrifuge tube. We recommend using a 1.5-ml tube for < 50 mg of tissue and a 12-ml 2059 tube for > 50 mg of tissue.

Immediately homogenize the tissue using a motor-driven homogenizer that is compatible with your microcentrifuge tube. Start at a low speed and gradually increase speed until the homogenate appears smooth with no visible particulate matter (~15-30 seconds). Keep foaming to a minimum by adjusting the speed. Note: Thorough homogenization is required for maximum mRNA yield.

Transfer the tube to a microcentrifuge and spin at maximum speed for 5 minutes at room temperature.

Incubate the sample at 45°C in a water bath or incubator for 10 minutes. While the sample is incubating, proceed to Prewash and Binding Procedure below.

When following the procedures in this section, be careful to never let the beads dry out.

Prewash and Binding Procedure

While the sample is incubating (above), begin the procedure below to wash the beads and bind the sample:

Thoroughly resuspend the FastTrack® MAG Beads by pipetting them gently up and down. Transfer the amount of resuspended beads specified in the table to an RNase-free microcentrifuge tube.

Insert the microcentrifuge tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), pipette the liquid out of the tube and discard, and immediately add the volume of Wash Buffer W7 specified in the table. Important: Do not let the beads dry out.

Remove the tube from the magnetic separator and resuspend the beads in the wash buffer by pipetting gently up and down.

When the incubation time of the sample has ∼2 minutes left (Step 5, Preparing the Tissue Sample, previous page), repeat the wash procedure in Steps 2 and 3 above one time, and then proceed to Step 5 below.

Insert the tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), pipette the liquid out of the tube and discard, and immediately add the sample from Step 5, previous page, and the heated Binding Buffer B6 from Step 1.

Remove the tube from the magnetic separator and resuspend the beads completely in the solution by pipetting up and down.

Cap the tube and place in the heat block or thermal cycler. Incubate at 65–70°C for 2–5 minutes.

Transfer the tube to a rotator and rotate the sample for 10 minutes at room temperature. Proceed to Wash and Elution Procedure.

Wash and Elution Procedure

Insert the tube from Washing and Binding Procedure, Step 8, previous page, in the magnetic separator. Visually inspect the beads until they are clearly separated (∼2–5 minutes). Note: Beads may take longer to separate depending on the viscosity of the sample and the abundance of non-sheared genomic DNA.

When the beads are separated, remove and save the supernatant. Note: For rare samples, if your yield is low, you may want to preserve this supernatant and attempt to perform a separate isolation with it.

To the beads, immediately add the volume of Wash Buffer W6 specified in the table

Remove the tube from the magnetic separator and resuspend the beads in the wash buffer by pipetting gently up and down.

If you are using > 50 mg tissue and/or performing downstream RT-PCR: Add 1 μl (1 unit) of DNase I, Amplification Grade, per 100 μl of Wash Buffer W6 to the tube. Mix by pipetting gently up and down and incubate at 25°C for 5–10 minutes.

Reinsert the tube in the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), remove the wash buffer from the tube and discard. Be careful to completely remove the wash buffer.

To the beads, immediately add the volume of Wash Buffer W7 specified in the table.

Remove the tube from the magnetic separator and resuspend the beads by pipetting gently up and down.

Reinsert the tube in the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), remove the wash buffer from the tube and discard. Be careful to completely remove the wash buffer.

Repeat Steps 7–9 using Wash Buffer W7 two more times, then proceed immediately to Step 11.

If you started with < 50 mg of tissue, immediately add 5–20 μl of RNase-free water. If you started with > 50 mg of tissue, immediately add 20–50 μl of RNase-free water.

Remove the tube from the magnetic separator and thoroughly resuspend the beads by pipetting gently up and down. Incubate at 37°C for 2–5 minutes. Use the longer incubation time for larger samples.

Insert the tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), remove and save the supernatant. Important: The supernatant contains the isolated mRNA. Do not discard.

Repeat Steps 11–13, using 5 μl of RNase-free water for < 50 mg of tissue and 10 μl of RNase-free water for > 50 mg of tissue. Again, save the supernatant.

Combine the supernatants from Steps 13 and 14. This is your isolated mRNA. Store mRNA at -80°C.

The mRNA quality and yield may be determined and spectrophotometry and agarose gel electrophoresis.

Determining mRNA Yield and Quality

The following general protocol may be used to calculate the yield of mRNA using A260 absorbance:

Aliquot 2 μl of the isolated mRNA into a clean UV cuvette and add 198 μl of TE Buffer for a 1:100 dilution.

Blank a UV/visible spectrophotometer using TE Buffer, and then measure the sample at 260 nm.

The A260 reading should fall within the standard specification for the spectrophotometer (typically 0.01–1.0 OD). If it falls outside this range, adjust the dilution and rescan. If the A260 reading is too low, use a lower dilution; if it’s too high, use a higher dilution.

The quality of the isolated mRNA may be determined by agarose gel electrophoresis. The mRNA appears as a smear from 500 bp to 8 kb, with the highest intensity between ∼1–3 kb. Any contaminating ribosomal RNA will appear as bands within the mRNA smear. These bands should be faint (< 20% higher intensity than the rest of the smear).

Check the quality of the purified total RNA on a gel. The 28S band should have a higher intensity than the 18S band, and the 18S band should have a higher intensity than the 5S band. If you see an obvious smear at low molecular weight (between 5S to 18S rRNA), it indicates that the total RNA is partially degraded.

Samples have a low abundance of RNA

Use more starting material.

mRNA is contaminated with rRNA

Poor removal of rRNA during wash steps

Wash the beads 1 or 2 more times.

Perform the full procedure again, using the isolated mRNA from the first procedure.

mRNA is contaminated with genomic DNA

Poor removal of genomic DNA during the wash steps

Use the optional DNase I digestion step to remove genomic DNA contamination. Be sure to use Amplification Grade DNase I.

Viscous cell lysate

Sample is too large (mRNA will bind to oligo(dT) beads if the viscosity is reduced) Reduce the sample size, or split the sample.

Sample contains large amounts of DNA

Reduce the sample size, or split the sample.

Split the sample as above and shear the DNA thoroughly using a 21-guage needle on a 1-ml syringe.